Regeneration of plants is obligatory and often limiting stage of such biotechnologies as cell selection, genetic engineering, somatic hybridization, generation of haploid and diploid plants, microclonal proliferation.
Microclonal proliferation is a method of vegetative proliferation of plants by activation of dormant buds, induction of formation of new (adventitious) buds or callus tissues with subsequent generation of plans from them. Apart from proliferation, this method also provides partial resistance of planting material to fungal, bacterial and virus diseases. To date all of elite planting material of potato, berry, vegetable and decorative cultures is produced by using microclonal proliferation. Clonal proliferation of lignosa is of especially great interest [Ahuja, M. R. and W. J. Libby (Eds.). 1993. Clonal Forestry I: Genetics and biotechnology, Clonal Forestry II: Conservation and application. Springer-Verlag. Berlin]. When plans are produced from cultivated tissues, the two basic problems arise: how to increase amount of regenerants produced and how to implant them. To solve the first problem, nutrient medium is supplemented with growth regulators possessing cytokinin activity. However, shoots formed are “overfed” with cytokinin and, therefore, they take roots badly. Offered programs of gradual exclusion of cytokinins, addition of different growth regulators with auxin action demand a lot of time and not always produce a positive result. Long-term cultivation of plant tissues in vitro can cause undesirable mutations. To increase efficiency of clonal proliferation, more sophisticated regulation of plant regeneration is required (Tree Physiol. 2000 August; 20(14):921-8; Curr Opin Biotechnol. 2000 June; 11(3):298-302; Methods Mol Biol. 1999; 111:127-34).
Generation of haploid plants is applied to fix heterosis effect in hybrids. Upon usual proliferation by seeds, the optimum combination of parental genomes quickly collapses and, therefore, heterosis is not preserved longer 1-2 generations. To fix hybrid genotypes, haploid plants are produced from pollen of best plants F2 and then duplication of chromosomes is achieved. Dihaploid plants produced can blossom normally and produce seeds. Since chromosomes in pair are identical, properties of such hybrid remain constant upon proliferation. To produce haploids, anthers or isolated pollen grains at early stages of development are introduced into in vitro culture and somatic embryogenesis or callusogenesis is induced. Probability of haploid plant generation does not exceed 5-10% of a number of planted anthers and significantly depends on a genotype of initial plants. Usually albinism can often be observed among regenerants. Upon production of haploids, the main goal is reprogramming of pollen grains from normal development (germination) to somatic embryogenesis. In this case, exogenetic regulation by phytohormones is ineffective. Some signals changing synthesis or activity of endogenous growth regulators are necessary.
Somatic hybridization is a process of fusion of isolated protoplasts separated from plant cells. Somatic hybridization allows to overcome noncrossing barriers upon distant hybridization, enables to generate unique combinations of nuclear and plastomic genomes of parents. Method of somatic hybridization is based on fusion of protoplasts induced by different ways followed by regeneration of cell wall at special nutrient media, hybrid cell division and callus formation. Then callus is used for plant regeneration. The most simple stage of this method is isolation and fusion of protoplasts, it is more difficult to achieve hybrid cell division, and the most difficult stage is assumed to be regeneration of plants from calli formed.
Isolation of plant tissues and their cultivation under artificial conditions induce genetic variation, which can also be shown in plants generated from cultivated cells. This phenomenon named somaclonal variation can be used, along with induced mutagenesis, for increase of genetic variation of crops in agriculture. Using selective nutrient media, it is possible to select cells with the given attributes directly in in vitro culture-to produce plants. Cell selection is mainly applied to increase resistance of plants to diseases and pests, and also to such adverse environmental factors as drought, salting, extreme temperatures, flooding etc. The selective systems aimed at increasing both specific and nonspecific resistance have been developed.
The combination of traditional and cell selection methods has already allowed to produce improved forms and new varieties of tomatoes, sugar cane, rice, barley, potato, spinach, fodder grasses and some other kinds of plants possessing resistance to biotic and abiotic stresses and high productivity [Karp A. Somaclonal variation as a tool for crop improvement//Euphytica, 1995, v. 85, p. 295-302].
Genetic engineering offers novel opportunities for generation of new varieties. Crops in agriculture tolerant to insects-pests, herbicides, adverse climatic conditions have been produced by introduction of genes derived from bacteria or plants. Plants with increased content of protein and essential amino acids, improved oil quality etc have been generated [Ya. I. Bur'yanov. Advances in Plant Gene Engineering Biotechnology.//Russian journal of plant physiology (Fiziologiya rastenii)-1999—v. 46—No 6—p. 930-944]. Almost all methods of genetic transformation comprise stages of cultivation of tissues in vitro and regeneration of plants from transgenic cells.
Thus efficiency of application of all cell biotechnologies depends on a possibility to produce a plant from cultivated cells (Plant Cell Culture Protocols. Second edition. V. M. Loyola-Vargas and F. Vazquez-Flota (eds). Humana Press, Mexico, 2005, 416 pp.; Plant Tissue Culture. 100 years since Gottlieb Haberlandt. M. Laimer and W. Rucker (eds.). Springer, Wien N.Y., 2003, 260 pp.).
The basic obstacle in wide use of cell cultures in selection is low regeneration ability of many lines and varieties. For example, in cotton, variety Coker and its derivatives are highly capable for morphogenesis, whereas a majority of other varieties has lowered or zero regeneration potential (Theor Appl Genet. 2004 August; 109(3):472-9.). Production of regenerants in some leguminous plants including such important plant as soya is a serious problem (Planta. 2004 October; 219(6):1042-9). In grain cereals, only cell cultures produced from embryos are really capable for morphogenesis (Vasil V., Chin Yu. L., Vasil I. K. Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)//Protoplasma, 1985, v. 127, p. 1-8). A possibility of plant regeneration strongly depends on genotype (J Exp Bot. 2005 July; 56(417):1913-22.). The majority of commercial corn hybrids is characterized by low morphogenetic potential (Phillips R. L., Somers D. A., Hiberd K. A. Cell/tissue culture and in vitro manipulation. In: Corn and Corn Improvement—Agronomy Monograph 18. (Sprague G. F., Duddley J. W. eds) Am. Soc. of Agronomy, Madison, Wis., 1988, p. 345-387).
It is important that regeneration of plants from undifferentiated tissues and cells is a key stage upon production of any genetically modified plants.
Choice of Explant Tissue
The abilities of different plant tissues to form morphogenetic callus in in vitro culture are distinctive. This feature is most pronounced in monocotyledons. Meristematic tissues: unripe germs, inflorescences, meristem in nodes of tillering and in bases of leaves are characterized by highest regeneration potential. Differentiated tissues of leaves or roots have low ability to callusogenesis. Therefore, if plants are to be produced from cultivated cells, it is necessary to produce callus from competent tissues (Phillips R. L., Somers D. A., Hiberd K. A. Cell/tissue culture and in vitro manipulation. In: Corn and Corn Improvement—Agronomy Monograph 18. (Sprague G. F., Duddley J. W. eds) Am. Soc. of Agronomy, Madison, Wis., 1988, p. 345-387).
Variations of Hormonal Content of Nutrient Medium
The basic way of switching cells from unorganized growth to differentiation is change in concentration and ratio of hormones. To regenerate many plant species, it is necessary to increase concentration of cytokinins in a medium (Adv Biochem Eng Biotechnol. 2001; 72:157-82.). Ratio and concentration of phytohormones in a nutrient medium optimum for morphogenesis are species- and even variety-specific; therefore anew morphogenesis conditions should be selected upon introduction of new varieties or species of plants. In some cases a probability of morphogenesis increases upon replacement of traditionally used natural and synthetic phytohormones with compounds of other chemical nature but possessing a hormonal activity. However upon screening of a large amount of varieties, different authors have shown that stimulation is also variety-specific, and in case of success frequency of plant regeneration has increased no more than 20% [Wilkinson, Thompson, 1987], [Ignatova et al., 1993; Dias S., Dolgikh Y. I. Role of physiological factors in increase of efficiency of plant regeneration from cultivated maize tissues. Biotechnology (in Russian), 1997, No 11-12, p. 32-36].
Ethylene clearly inhibits callus regeneration. Addition of ethylene precursors 1-aminocyclopropane-1-carboxylic acid or aminoethoxyvinylglycine to a nutrient medium for callus initiation caused significant reduction of frequency of embryogenic callus formation and decrease of a number of produced regenerants by 68% [Songstad D. D., Duncan D. R., Widholm J. M. Effect of 1-aminocyclopropane-1-carboxylic acid, silver nitrate and norbornadiene on plant regeneration from maize callus cultures//Plant Cell Rep., 1988, v. 7, p. 262-265.] [Vain P., Flament P., Soudain P. Role of ethylene in embryogenic callus initiation and regeneration in Zea mays L.//J. Plant Physiol., 1990, v. 135, p. 537-540]. Inclusion of compounds inhibiting physiological effect of ethylene-norbornadiene or silver nitrate, into the medium, on the contrary, stimulated embryogenic callus formation and raised regeneration efficiency in a majority of tested genotypes by 15-20% [Vain P., Flament P., Soudain P. Role of ethylene in embryogenic callus initiation and regeneration in Zea mays L.//J. Plant Physiol., 1990, v. 135, p. 537-540]. [Hoisington D. A., Bohorova N. E. Towards the production of transgenic tropical maize germplasm with enhanced insect resistance. In: Current issues in Plant Molecular and Cellular Biology (Terzi M., Cella R., Falavigna A. eds.), Kluwer Acad. Publishers., Netherlands, 1995, p. 327-221].
In some cases, addition of abscisic acid to the medium enhances morphogenesis. In wheat, addition of abscisic acid at micromolar concentration inhibited premature germination of isolated embryos and stimulated embryogenic callus formation [Brown C., Brooks F. J., Pearson D., Mathias R. J. Control of embryogenesis and organogenesis in immature wheat embryo callus using increased medium osmolarity and abscisic acid//J. Plant Physiol., 1989, v. 133, p. 727-733; Carman J. G. Improved somatic embryogenesis in wheat by partial simulaton of the in-ovulo oxygen, growth-regulators and desiccation environments//Plants, 1988, v. 175, p. 417-424; I. F. Shayakhmetov, F. M. Shakirova. Somatic Embryogenesis in Wheat Cell Suspension Cultures in the Presence of Abscisic Acid.//Russian journal of plant physiology (Fiziologiya rastenii), 1996, v. 43, p. 101-103]. In tissue culture of wild turnip (Brassica napus) and maize, effect of abscisic acid on regeneration of plants was positive for some varieties but negative for others [Raldugina G. N., Sobolkova G. I. Genotypic differences upon abscisic acid action on callus cultures Brassica napus L.//Russian journal of plant physiology (Fiziologiya rastenii), 1994, v. 41, p. 702-706; Yu. I. Dolgikh, T. N. Pustovoitova, N. E. Zhdanova. Hormonal regulation of somatic embryogenesis on maize. In Phytohormones in Plant Biotechnology and Agriculture, Proceedings of NATO-Russia Internation Workshop, Kluwer Academic Publishers, 2003, p. 243-247].
Rather often, none of the media used provide production of plants-regenerants [Armstrong C. L., Romero-Severson J., Hodges T. K. Improved tissue culture response of an elite maize inbred through backross breeding, and identification of chromosomal regions important for regeneration by RFLP analysis//Theor. Appl. Genet., 1992, v. 84, p. 755-762].
Inclusion of Individual Amino Acids into Medium Content
Amplification of morphogenetic potential has been observed in some cultures upon addition of some amino acids to medium. For instance, agrinine-containing media are used for wheat, proline-containing media are used for maize [Salmenkallio M, Sopanen T. Amino Acid and Peptide Uptake in the Scutella of Germinating Grains of Barley, Wheat, Rice, and Maize//Plant Physiol. 1989 v. 89 p. 1285-1291]. It was recommended to use proline at high concentrations for increase of frequency of formation of friable embryogenic callus with prolonged ability of regeneration [Duncan D. R., Williams M. E., Zehr B. E., Widholm J. M. The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes//Planta, 1985, v. 165, p. 322-332]. According to Rapela's data, inclusion of 400 mg/L proline into medium resulted in 2-3-fold increase of frequency of embryogenic callus formation and plant regeneration [Rapela M. A. Organogenesis and somatic embryogenesis in tissue culture of Argentine maize (Zea mays L.)//J. Plant Physiol., 1985, v. 121, p. 119-122]. In other work, 20 mM proline caused increase in a number of regenerants per explant from 2.8 up to 7.6 [Kamo K. K., Becwar M. R., Hodges T. K. Regeneration of Zea mays L. from embryogenic callus//Bot. Gaz., 1985, v. 146, p. 327-334]. The data on application of asparagine are ambiguous: along with stimulation of somatic embryogenesis in cultivated maize tissues [Lupotto E. In vitro culture of isolated somatic embryos of maize (Zea mays L.)//Maydica, 1986, v. 31, p. 193-201; Morocz S., Donn G., Nemeth J., Dudits D. An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture//Theor. Appl. Genet., 1990, v. 80, p. 721-726] its oppression has also been observed [Kamo K. K., Becwar M. R., Hodges T. K. Regeneration of Zea mays L. from embryogenic callus//Bot. Gaz., 1985, v. 146, p. 327-334]. The mechanism of influence of proline or asparagine on ability of morphogenesis has not been described.
Influence of Oligosaccharides
Oligosaccharides are formed during disintegration of plant cell walls. For instance, xyloglucan pentasaccharide 1.5-2-fold enhanced morphogenesis in wheat tussue culture [Pavlova Z. N., Ash O. A., Vnuchkova V. A., Babakov A. V., Torgov V. I., Nechaev O. A., Usov A. I., Shibaev V. N. Biological Activity of a Synthetic Pentasaccharide Fragment of Xyloglucan//Plant Sci. 1992 V. 85. P. 131-134], and trimerous oligosaccharide promoted somatic embryogenesis in cotton [Yu. I. Dolgikh, E. Yu. Shaikina, A. I. Usov, V. N. Shibaev, V. V. Kuznetsov. The Trisaccharide Fragment of Xyloglucan as a Regulator of Plant Morphogenesis//Doklady Akademii Nauk. 1998. v. 360. p. 417-419].
Electrostimulation of Morphogenesis
Passing the weak (1-2 μA) constant electric current through calli was shown to stimulate shoot regeneration. 1 to current resulted in increase of tobacco callus weight by 70% and 5-fold increase of a number of shoots formed [Goldsworthy A. The electric compass of plants//New Sci., 1986, No 1, p. 22-23]. In wheat callus, two-fold amplification of rhizogenesis and shoot formation were shown whereas they were entirely absent in the control [Rathore K. S., Goldsworthy A. Electrical control of shoot regeneration in plant tissue culture//Bio/technology, 1985, v. 3, p. 1107-1109]. Stimulation of somatic embryogenesis in lucerne protoplast culture [Dijak M., Smith D. L., Wilson T. J., Brown D. C. W. Stimulation of direct embryogenesis from mesophyllprotoplasts of Medicago sativa//Plant Cell Rep., 1986, v. 5, p. 468-470], and activation of shoot formation in cabbage, poplar, maize [Wang X., Wang Q., Song M., Zheng E. Effect stimulation with weak electric currents on in vitro culture of cabbage//Acta Bot. Sinica, 1993, v. 35, Suppl., p. 66-70; Dutta R. Studies on the mechanism of electrically induces growth and differentiation in plants in vitro: the cytomorphological profile. Abstr. VIII Intern. Congr. “Plant Tissue and Cell Culture”, Florence, Italy, 1994, p. 49; Kitlaev G. B., Dolgikh Yu. I., Butenko R. G. Physiological action of electric current on maize cell culture in vitro. Doklady Akademii Nauk, 1994, v. 335, No 3, p. 393-395] were also found out. Electric current-induced stimulation of plant regeneration is independent of plant variety or species though quantitative variations are possible. Application of said method of increase of frequency of plant regeneration is limited by necessity to have rather complex technique and small throughput.